PROJECT SUMMARY The homeodomain transcription factor PROX1 is necessary and sufficient for induction and maintenance of lymphatic endothelial cell identity. It not only plays an essential role in the initial lymphatic reprogramming, expansion, maturation and maintenance, but also directs formation of luminal and lymphovenous valves. Although Prox1 has been intensively studied for its function, structure, and regulation, it remains unclear how such numerous developmental and environmental signals are efficiently incorporated to and regulate Prox1. The objective of this study is to gain a detailed mechanistic understanding of how lymphangiogenic signals triggered by growth factors and inflammatory cytokines are transduced to Prox1 protein in the form of phosphorylation, modulate its properties, and eventually orchestrate lymphatic development and function. We hypothesize that the RAF-ERK-RSK signal cascade mediates various lymphangiogenic signals and phosphorylates PROX1 at S79, and that this modification significantly alters the biological properties of Prox1 during lymphatic development and function. To address these hypotheses, we propose to study of the impact of phospho-S79 to the behaviors of PROX1, and to elucidate the regulation of lymphangiogenesis by the ERK-RSK2-PROX1 (S79) axis under the physiological and pathological conditions. Finally, we will generate mutant mouse models that allow a tissue- specific, conditional replacement of the endogenous wild type Prox1 with its phospho-mutants. Using these animal models, we will study of the impact of PROX1 S79 phospho-mutation to lymphatic development and function in health and disease. In summary, the proposed study will define how Prox1 S79 phosphorylation, as a biomarker of activated lymphatic vessels, regulates physiological and pathological lymphangiogenesis. The outcome of this study will not only deliver a significant impact on our current understanding of the functional mode of PROX1 as the master regulator of the lymphatic system, but also offer broader insights into vascular development and function.